PubMed Health⌕ Search

Biomedical subjects

M Brüss

Publications and source records attributed to M Brüss.

At least 37 records · Page 2Linked to original sources
PubMed1999

Presynaptic imidazoline receptors. New developments in characterization and classification.

Presynaptic imidazoline receptors (IRs) which inhibit norepinephrine (NE) release from sympathetic nerve endings have been identified in cardiovascular tissue of man, rabbit, rat, and guinea pig. They do not belong to one of the classical presynaptic inhibitory receptor classes such as alpha 2-adrenoceptors or H3 histamine receptors, and there is also no relation to I1- and I2-imidazoline binding sites. Segments of human right atrial appendages preincubated with [3H]NE were used to determine unknown pharmacologic properties of the presynaptic IRs. In the presence of 1 microM rauwolscine, S23230, the (-)-enantiomer of the racemic oxazoline derivative S22687 (5-(2(methyl-phenoxy-methyl)-1,3-oxazoline-2-yl)amine) exhibited low potency in inhibiting the electrically evoked [3H]NE release (pIC30% = 4.96), whereas the (+)-enantiomer S23229 and the racemate S22687 were ineffective. The IR-mediated inhibitory effect of the imidazoline BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline) and the guanidine aganodine on evoked [3H]NE release from sympathetic nerves in human atrial appendages was counteracted by rauwolscine (with very low potency) and by the cannabinoid CB1-receptor antagonist SR141715A (N-[piperdin-1-yl]-5-[4-chlorophenyl]-2,4-dichlorophenyl]-4- methyl-1H-pyrazole-3-carboxamide). The inhibitory effect of moxonidine on evoked [3H]NE release (which is exclusively mediated via activation of alpha 2-autoreceptors) was antagonized with high potency by rauwolscine, but not by SR141716A. The cannabinoid CB1 receptor agonists CP55,940([(-)-Cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl] -trans-4- (3-hydroxy-propyl)cyclohexane]) and anandamide inhibited evoked [3H]NE release. Inhibition by CP55,940 and anandamide was abolished by 1 microM SR141716A as well as by 30 microM rauwolscine. In radioligand binding experiments on membranes from human atrial appendages (alpha 2- and sigma-binding sites were masked), cannabinoid receptor ligands and IR agonists displaced the radiolabeled guanidine derivative [3H]DTG (1,3-di-o-tolyguanidine, an agonist at presynaptic IRs) from its binding sites. Comparison of the potencies of these drugs determined in the competition experiments with [3H]DTG with those in inhibiting NE release via activation of the presynaptic IRs in the same tissue revealed a correlation. The present results suggest, e.g., that the presynaptic IRs may have certain binding domains in common with presynaptic cannabinoid receptors or that both receptors are different proteins which interact with each other in an unknown manner.

Animals↗
PubMed1999

Tourette syndrome and the norepinephrine transporter gene: results of a systematic mutation screening.

Tourette syndrome (TS) is a complex inherited neuropsychiatric disorder characterized by multiple motor and phonic tics. Involvement of central norepinephrine mechanisms is suggested by central norepinephrinic hyperactivity in patients with TS and by the therapeutic effects of the presynaptic alpha2-adrenergic agonist clonidine. The norepinephrine transporter gene (NET) was systematically screened by single-strand conformation analysis for genetic variants, including the whole coding region and adjacent exon-intron boundaries in 43 patients with TS and 46 healthy controls. We detected 12 DNA sequence variants, among them four missense mutations (Val69Ile, Thr99Ile, Va1245Ile, and Gly478Ser). The observed missense mutations may alter conformational rearrangements during gating of the transporter, assembly of subunits, and norepinephrine-specific uptake affinity. Allele frequency and genotype distribution of the genetic variants showed no differences between TS patients and controls. No mutation of likely functional significance was found that distinguished TS patients from healthy controls, indicating that genetic variants of the NET gene are not causally related to Tourette syndrome.

Adolescent↗
PubMed1999

Pharmacological differences and similarities between the native mouse 5-HT3 receptor in N1E-115 cells and a cloned short splice variant of the mouse 5-HT3 receptor expressed in HEK 293 cells.

Human embryonic kidney (HEK) 293 cells were stably transfected with the cDNA encoding the short splice variant of the mouse 5-HT3 receptor (m5-HT3A(b); isolated by RT-PCR from NG108-15 cells) and its pharmacological properties were compared with those of the native 5-HT3 receptor of the mouse neuroblastoma cell line N1E-115. The m5-HT3A(b) receptor of N1E-115 cells differs from that isolated from NG108-15 cells by one amino acid (Val instead of Ile) at position 52 of the amino acid sequence. Both radioligand binding studies with the selective 5-HT3 receptor antagonist [3H]GR65630 (3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)-1-propanone) and functional experiments by measurement of [14C]guanidinium influx evoked by 5-HT in the absence and presence of 10 microM substance P were carried out. Binding of [3H]GR65630 to the recombinant receptor in HEK 293 cells and the native receptor in N1E-115 cells was specific and of high affinity (Kd 4.4 and 3.0 nM, respectively) and characterized by Bmax values of 875 and 1414 fmol/mg protein, respectively. At 10 nM [3H]GR65630, specific binding was inhibited by the selective 5-HT3 receptor antagonist ondansetron (Ki 11 and 42 nM, respectively) and by 5-HT (Ki 294 and 563 nM, respectively). In the transfected HEK 293 cells, 5-HT induced an influx of [14C]guanidinium both in the absence (pEC50 5.7) and presence of substance P (pEC50 6.6,) which was counteracted by 0.3 microM ondansetron; in the N1E-115 cells, 5-HT also evoked [14C]guanidinium influx in the absence (pEC50 6.0) and presence of substance P (pEC50 6.0). Both in transfected HEK 293 cells and in N1E-115 cells, the 5-HT receptor ligand RS-056812-198 ((R)-N-(quinuclidin-3-yl)-2-(1-methyl-1 H-indol-3-yl)-2-oxo-acetamide; in the presence of substance P) induced an influx of [14C]guanidinium (pEC50 9.8 and 8.7, respectively) with a maximum of about 70 and 30% of the maximum response to 5-HT, respectively. 5-HT (in the presence of substance P)-induced [14C]guanidinium influx was inhibited by the imidazoline BDF 6143 (4-chloro-2(2-imidazolin-2-ylamino)-isoindoline; pIC50 4.9 and 5.3, respectively) and by the sigma-site ligand (+/-)-ifenprodil (pIC50 5.0 and 5.2, respectively). In conclusion, most of the drugs exhibited practically identical properties at both the recombinant m5-HT3A(b) receptor in HEK 293 cells and the native m5-HT3 receptor of N1E-115 cells. However, the recombinant receptor had a higher affinity for ondansetron, and the potency of 5-HT in inducing cation influx through the recombinant, but not through the native receptor, was increased by substance P. RS-056812-198 was a 10-fold more potent partial agonist at the recombinant than at the native receptor. These differences may be due to cell-specific post-translational modifications of the 5-HT3 receptor protein in the two cell lines, to the expression of other subunits in addition to the m5-HT3A(b) receptor in N1E-115 cells and/or to the difference in the amino acid sequence at position 52 of the short splice variants of the m5-HT3 receptors expressed in the two cell lines.

Alternative Splicing↗
PubMed1999

Inhibition by steroids of [14C]-guanidinium flux through the voltage-gated sodium channel and the cation channel of the 5-HT3 receptor of N1E-115 neuroblastoma cells.

Effects of some naturally occurring steroids and synthetic analogues on the cation flux through the cation channel of the 5-HT3 receptor and the voltage-gated and tetrodotoxin-sensitive sodium channel were studied in N1E-115 mouse neuroblastoma cells by measuring the 2-min influx of the organic cation [14C]-guanidinium. The cation fluxes in intact cells were either induced by 2 min exposure of the cells to 5-hydroxytryptamine (5-HT, 100microM) or to veratridine (1 mM). Influx of [14C]-guanidinium through both channels was concentration-dependently inhibited by all compounds studied. The rank order of potency for inhibition of the 5-HT3 receptor-induced cation flux was clomiphene approximately/= cyproterone acetate > estradiol > progesterone approximately/= allotetrahydrodeoxycorticosterone > alfaxalone approximately/= testosterone > aldosterone > dexamethasone. With the exception of dexamethasone and testosterone, which were more potent at the voltage-dependent sodium channel, and progesterone and testosterone, which were about nearly equipotent in inhibiting both cation channels, the steroids were twofold (alfaxalone, allotetrahydrodeoxycorticosterone) to 107-fold (cyproterone acetate) more potent at the 5-HT3 receptor channel than at the voltage-gated sodium channel. The potencies of the steroids (and the synthetic analogues) for inhibition of the 5-HT3 receptor-induced [14C]-guanidinium influx were correlated with their lipophilicity (log P values). A similar correlation between log P values and pIC50 values for the steroid-induced inhibition of the veratridine-evoked cation influx through the voltage-gated sodium channel was only found when cyproterone acetate (a compound with extremely low inhibitory potency at this channel) was not included in the regression analysis. The results indicate that both the 5-HT3 receptor channel and the voltage-gated sodium channel are targets for steroids. The relationship between most of the compounds in inhibiting both cation channels and lipophilicity is compatible with a common mechanistic principle in steroid-induced inhibition of the two channels, i.e. a nonspecific hydrophobic interaction with certain membrane lipids in the neighbourhood of the two channels.

Animals↗
PubMed1999

Molecular cloning and functional expression of the mouse dopamine transporter.

The full coding region of the murine dopamine transporter (mDAT) cDNA was cloned by PCR with a sense primer derived from the partial mDAT gene sequence and an antisense primer deduced from the rat dopamine transporter cDNA. The mDAT cDNA encodes a typical member of the family of Na+- and Cl--dependent neurotransmitter transporters with 99.2; 93.4 and 85.4% amino acid identity to the rat, human and bovine DATs, respectively. Functional expression of the mDAT cDNA in transiently transfected human embryonic kidney (HEK293) cells exhibited the typical pharmacological features of a dopamine transporter. [3H]dopamine uptake through the mDAT was inhibited with high potency by GBR12909 (IC50 = 5.2 nM) and not significantly affected by 100 nM desipramine. [3H]dopamine uptake also was inhibited through increasing concentrations of dopamine (IC50 = 0.93 microM) or 1-methyl-4-phenylpyridinium (MPP+; IC50 = 13.2microM).

Animals↗
PubMed1999

Molecular cloning, functional characterization and genomic organization of four alternatively spliced isoforms of the human organic cation transporter 1 (hOCT1/SLC22A1).

In this study we report the cloning of four human OCT1 (hOCT1/SLC22A1) isoforms: a long form, hOCT1G/L554, and three shorter forms (hOCT1G/L506, hOCT1G483 and hOCT1G353). All four variants could be identified in the human glioma cell line SK-MG-1, whereas only two isoforms (hOCT1G/L554 and hOCT1G/L506) were found in human liver cDNA. The hOCT1G/L554 represents the full length hOCT1 since the sequence of this clone is more than 99% identical to previously cloned hOCT1 cDNAs. Elucidation of the gene structure of human OCT1 demonstrated that the other isolated isoforms are alternatively spliced variants. The hOCT1 gene consists of 7 exons and 6 introns. When stably expressed in human embryonic kidney (HEK293) cells, only the full length hOCT1 cDNA mediated decynium-22 (D22)-sensitive uptake of tritiated 1-methyl-4-phenylpyridinium ([3H]-MPP+).

1-Methyl-4-phenylpyridinium↗
PubMed1999

The human desipramine-sensitive noradrenaline transporter and the importance of defined amino acids for its function.

1. This article gives a short overview of the physiology, pharmacology and the molecular biology of the human Na+/Cl(-)-dependent noradrenaline transporter (hNAT) and its gene. 2. Furthermore, naturally occurring variants of the hNAT are described and new results obtained through site-directed mutagenesis of the hNAT are presented, which increase our understanding about structural domains and amino acids critically involved in substrate, cosubstrate and inhibitor binding to the hNAT.

Adrenergic Uptake Inhibitors↗
PubMed1999

Modified ligand binding to the naturally occurring Cys-124 variant of the human serotonin 5-HT1B receptor.

A substitution of phenylalanine by cysteine in position 124 is the only known naturally occurring variant of the human 5-HT1B (h5-HT1B) receptor. The present study was designed to evaluate the potential influence of the Cys-124 variant on pharmacological properties of the receptor and to test for an involvement of the mutation in the genetic predisposition to schizophrenia or bipolar affective disorder. Binding of [3H]5-carboxamidotryptamine ([3H]5-CT) and its competition with serotonin receptor agonists and antagonists were determined in COS-7 cells transfected with the wild-type or the variant h5-HT1B receptor cDNA. In saturation experiments with [3H]5-CT, the maximum binding (Bmax) of the variant receptor was approximately 30% of the wild-type receptor. In competition experiments with 1 nM [3H]5-CT, the following serotonin receptor ligands exhibited a two to three times higher affinity for the mutant than for the wild-type receptor: dihydroergotamine, L-694,247, SB-216641, 5-CT, 5-HT, sumatriptan, RU24969 and methysergide (compounds listed at decreasing order of potency at the wild-type receptor). In contrast, the serotonin receptor antagonist ketanserin exhibited higher binding affinity for the wild-type than for the mutant h5-HT1B receptor and GR127939, (-)propranolol and BRL-15572 showed equal affinity for both types of receptor. Mutation screening of schizophrenic and bipolar patients revealed no relationship between the variant receptor and development of disease. In conclusion, our data suggest that the Cys-124 variant significantly affects the pharmacological properties of the h5-HT1B receptor. Carriers of the variant may exhibit differences in response to drugs acting on the h5-HT1B receptor or may develop side-effects to such agents.

Animals↗
PubMed1999

Comparison of the pharmacological properties of cloned rat, human, and bovine norepinephrine transporters.

The aims of this study were to characterize the recently cloned rat norepinephrine transporter (NET) in more detail and in particular to study possible species differences in its pharmacological properties compared with the human and bovine NETs. The study was carried out by measuring the uptake of [(3)H]norepinephrine in COS-7 cells expressing the NET after transient transfection with rat, human, or bovine NET cDNA. There were small but significant differences between the rat NET and the human or bovine NETs with respect to the affinities of sodium ions (greater for rat than for bovine) of the substrates norepinephrine, epinephrine, and 1-methyl-4-phenylpyridinium (greater for human than for rat), and of the inhibitor cocaine (greater for human and bovine than for rat), whereas the affinities of dopamine and of most inhibitors, including tricyclic antidepressants, showed no species differences. The fact that the affinities for some substrates, cocaine and sodium ions exhibited small but significant interspecies differences among the rat, human, and bovine NETs suggests that ligand recognition, the translocation process, and sodium ion dependence are influenced differentially by just a few amino acid exchanges in the primary sequences of the transporters. On the other hand, the lack of any major differences in the pharmacological properties of the rat, human, and bovine NETs in this study suggests that data obtained in previous studies on rat tissues and bovine cells can be extrapolated, in all except the most quantitative analyses, to the properties of the human NET.

1-Methyl-4-phenylpyridinium↗
PubMed1998

Genetic variation in human 5-HT receptors: potential pathogenetic and pharmacological role.

Mutation screening identified variants of h5-HT1A (Gly-22-Ser, Ile-28-Val, Arg-219-Leu), h5-HT1B (Phe-124-Cys), h5-HT2A (Thr-25-Asn, His-452-Tyr), h5-HT2C (Cys-23-Ser) and h5-HT7 (Thr-92-Lys, Pro-279-Leu) receptors. Screening of h5-HT1D, h5-ht1e, h5-ht1f and h5-ht5 receptor genes failed to detect any significant mutations. No differences in radioligand binding properties were observed between the h5-HT1A Ile-28-Val variant receptor (VR) and the wildtype receptor (WTR). Binding profiles of the h5-HT1A Gly-22-Val variant and the WTR were also very similar, but the 8-OH-DPAT-induced down-regulation and desensitization of the VR was attenuated. The h5-HT1B Phe-124-Cys variant leads to considerable changes in [3H]5-carboxamidotryptamine binding: Bmax was decreased and the affinity of various h5-HT1B ligands was modified (usually increased; e.g., in the case of sumatriptan). The h5-HT2A His-452-Tyr variant causes an alteration of the amplitude and timing of intracellular calcium mobilization in platelets from 452-His/452-Tyr heterozygous compared to 452-His/452-His homozygous individuals. Most, but not all, of the VRs listed above were examined for association with, e.g., bipolar depression and schizophrenia, yet no relation was observed. The most consistent finding was an association between a silent mutation (102T/C) in the h5-HT2A receptor gene and schizophrenia; this association may be explained by linkage disequilibrium with a functional variant in the regulatory region of the gene. Studies of the therapeutic response to clozapine produced no homogeneous results with respect to the pharmacogenetic significance of the various mutations in the h5-HT2A and h5-HT2C receptor genes.

Bipolar Disorder↗
PubMed1998

The human noradrenaline transporter gene contains multiple polyadenylation sites and two alternatively spliced C-terminal exons.

Sequencing downstream of the C-terminal exon 14 of the human noradrenaline transporter (hNAT) gene reveals 5 consensus polyadenylation signals, several adenylate/uridylate-rich elements (AREs) and a new C-terminal exon, designated as exon 15. The tandemly arranged polyadenylation sites are in good conformity with the 3.6- and 5.8-kb hNAT mRNA transcripts. Expression of the alternatively spliced C-terminal exon 15 is shown by RT-PCR. This alternative splicing event proposes additional hNAT mRNA species of 2.4-3 kb in size. Corresponding NAT transcripts are found by Northern analysis of human SKN and rat PC12 cell RNA. Sequence comparison of the hNAT gene to two bovine NAT cDNAs shows the interspecies conservation of this alternative splicing event, the close relationship of human and bovine NAT genes, and implicates a functional role for the transporters C-terminal domain.

Alternative Splicing↗
PubMed1998

A variant of the bovine noradrenaline transporter reveals the importance of the C-terminal region for correct targeting to the membrane and functional expression.

We have characterized a cDNA clone which encodes a variant (bNAT2) of the bovine noradrenaline transporter. This cDNA differs from the previously identified bovine noradrenaline transporter (bNAT1) in the sequence encoding part of the cytoplasmic-facing C-terminus and the 3'-untranslated region. The bNAT1 and bNAT2 cDNA clones are encoded by a 5.8 and 3.6 kb mRNA species respectively. The bNAT1 and bNAT2 proteins, which are identical apart from their C-terminal 31 and 18 residues, were stably expressed in HEK293 cells. Cells expressing bNAT1 showed a high level of desipramine-sensitive [3H]noradrenaline uptake activity, whereas no activity was present in bNAT2 cells. The bNAT1 and bNAT2 proteins were present as major 80 and 50 kDa species respectively. Cells expressing bNAT1 showed strong immunostaining of the plasma membrane, whereas bNAT2 was present in the endoplasmic reticulum/Golgi region. Treatment of membrane samples from bNAT1 cells with peptide N-glycosidase F resulted in the formation of a predominantly 50 kDa species, but little effect was observed after similar treatment of bNAT2 cell membranes. These results indicate that bNAT2 is retained in the endoplasmic reticulum and that the glycosylation of this variant differs from that of bNAT1. The characterization of bNAT2 and its comparison with bNAT1 highlight the importance of the cytoplasmic-facing C-terminus for the intracellular trafficking of neurotransmitter transporters.

Adrenal Medulla↗
PubMed1998

Ketamine interacts with the noradrenaline transporter at a site partly overlapping the desipramine binding site.

Effects of the intravenous anaesthetic ketamine on the desipramine-sensitive noradrenaline transporter (NAT) were examined in cultured bovine adrenal medullary cells and in transfected Xenopus laevis oocytes expressing the bovine NAT (bNAT). Incubation (1-3 h) of adrenal medullary cells with ketamine (10-300 microM) caused an increase in appearance of catecholamines in culture medium. Ketamine (10-1000 microM) inhibited desipramine-sensitive uptake of [3H]noradrenaline (NA) (IC50=97 microM). Saturation analysis showed that ketamine reduced Vmax of [3H]NA uptake without changing Km, indicating a non-competitive inhibition. Other inhibitors of NAT, namely cocaine and desipramine, showed a competitive inhibition of [3H]NA uptake while a derivative of ketamine, phencyclidine, showed a mixed type of inhibition. Ketamine (10-1000 microM) also inhibited the specific binding of [3H]desipramine to plasma membranes isolated from bovine adrenal medulla. Scatchard analysis of [3H]desipramine binding revealed that ketamine increased Kd without altering Bmax, indicating a competitive inhibition. In transfected Xenopus oocytes expressing the bNAT, ketamine attenuated [3H]NA uptake with a kinetic characteristic similar to that of cultured adrenal medullary cells. These findings are compatible with the idea that ketamine non-competitively inhibits the transport of NA by interacting with a site which partly overlaps the desipramine binding site on the NAT.

Adrenal Medulla↗
PubMed1998

Cloning and functional characterization of the human norepinephrine transporter gene promoter.

The norepinephrine transporter (NET) plays a critical role in brain norepinephrine homeostasis and is a target for antidepressants and drugs of abuse. We have analyzed the 5'-flanking regulatory region of the human NET gene (SLC6A2). Primer extension and 5' RACE revealed a single transcription start site, alternative splicing of exon 1 due to alternate splice donor usage, and a variable splice acceptor of intron 1. A TA-rich motif 35 bp upstream of the transcription start and several potential binding sites for transcription factors including a cAMP response element (CRE)-like motif are present in the 5'-flanking region. A 4.0-kb fragment, which had been fused to the luciferase reporter gene and transiently expressed in a NET+ cell line, displayed both constitutive and inducible promoter activity. Functional analysis by serial deletions revealed several clusters of cell-selective enhancer elements. Our findings indicate that (1) the NET gene promoter is active in NET-expressing cells and the information contained within approximately 4 kb of the 5'-flanking sequence is required to confer its cell-selective expression, (2) the expression of NET is regulated by a combination of positive cis-acting elements operating through a basal promoter defined by a TA-rich motif, and (3) the promoter responds to cAMP-dependent induction. Fusion of the human NET gene promoter to selected genes will facilitate their cell-selective expression in gene transfer strategies.

Animals↗
PubMed1997

The rat norepinephrine transporter: molecular cloning from PC12 cells and functional expression.

The rat norepinephrine transporter (rNET) cDNA from the PC12 pheochromocytoma cell line has been cloned by RT-PCR and characterized. The cDNA encodes an integral membrane protein consisting of 617 amino acids which contains twelve putative transmembrane domains, two potential N-glycosylation sites, two potential phosphorylation sites for protein kinase C and one phosphorylation site for casein kinase II. The nucleotide and deduced amino acid sequence shows a high level of homology to the human and the bovine norepinephrine transporter and less homology to the rat dopamine transporter (rDAT). Heterologous expression of rNET in HEK293 cells revealed that uptake of [3H]norepinephrine is sodium- and chloride-dependent and highly sensitive to the selective norepinephrine transporter inhibitors desipramine and nisoxetine. The cloned rNET cDNA provides the opportunity to investigate this transporter in heterologous expression systems and adds a new member to the family of sodium- and chloride-dependent neurotransmitter transporters.

Adrenal Gland Neoplasms↗